Combinatorial chemistry is a technology for simultaneously creating and rapidly screening a large number of different compounds r to identify useful compounds. Such peptide libraries can be used for the screening enzymatic substrates and inhibitors or cell binding peptides. Unlike the conventional synthetic way of handling one type of molecule at a time, combinatorial chemistry is an important tool for the discovery of new drug candidates, catalysts, and materials. Currently, several hundred peptide-based drugs have entered clinical phase testing or have already been commercialized, since peptides are considered as highly potent drug candidates due to their high specificity and low toxicity. Accordingly, the demand for the production of peptides in large quantities has also increased, and chemical synthesis methods using combinatorial chemistry play an important role.
Among various chemical synthesis methods, a solid phase peptide synthesis (SPPS) method, first described by Merrifield in 1963, has become a major breakthrough for the development of combinatorial chemistry due to its simplified reaction procedure and easy purification/isolation steps for the target products. The first amino acid is bounded to an insoluble support consisting of either resins or plastic pins and the desired sequence is built step by step by successive couplings of the appropriate protected amino acids. Reactions can be moved to completion by the use of excess reagents and repeated washings for purification. The methodology allows for automated peptide preparation relying on efficient chemistries without redundant and time consuming purification procedures. As a result, the synthesis of combinatorial libraries using solid phase chemistry has now become a routine strategy in the practice of drug discovery.
However, there are still a number of shortcomings associated with the use of solid phase chemistry, particularly in its analysis. Although mass spectrometry can offer high throughput analysis for combinatorial libraries, too many molecules do not have appropriate ionization properties for this technique to be universally applicable. Moreover, on-line monitoring of the multi-step synthesis using standard spectroscopic methods requires solubilization of the sample under study that is free from its solid support. Compound determination is thus usually achieved at the end of the synthesis since it is at this stage that the peptide is released from the insoluble support into solution. Utilizing such a cleavage and analysis strategy as a means of quality control and reaction monitoring presents several drawbacks. This type of compound assessment at an intermediate stage is destructive, as samples are consumed. Side-reactions with the cleavage reagents during this additional cleavage step may occur, leading to difficulties in the determination of peptides products by MS (mass spectrometry) due to the complicated mass spectra that are obtained.
Several reports (Michael C. F. et al., Bioorganic & Medicinal Chemistry Letters, Vol. 6, 979-982, 1996; Stephen C. M. et al., Tetrahedron Letters, Vol. 40, 2407-2410, 1999) have shown that matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) could be used to analyze selected Fmoc-protected amino acids or peptides bound to a solid-phase resin through a photolabile linker. Some other articles (Delphine M. et al., Journal of the American Society for Mass Spectrometry, Vol. 12, 1099-1105, 2001) reported that time-of-flight secondary ion mass spectrometry (TOF-S-SIMS) could be utilized to characterize analytes anchored to solid supports in a single step requiring no pretreatment of the sample. However, desorption and ionization in both MALDI-TOF and TOF-S-SIMS must be performed in high vacuum system. Monitoring solid phase peptide synthesis in real time for synthesis quality control is not possible with these kinds of techniques.
Development of a direct non- or minimally destructive on-line monitoring method would allow peptide solid-phase synthesis to be followed step by step for good quality control. The present disclosure provides such methods and addresses some of the limitations noted above.